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Establishment of Efficient Microinjection System in the Porcine Embryos
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  • Establishment of Efficient Microinjection System in the Porcine Embryos
  • Establishment of Efficient Microinjection System in the Porcine Embryos
저자명
Malaweera. Don Buddika Oshadi,Ramachandra. Sisitha,Wu. Jun-Bo,Oh. Seung-Kyu,Kim. Seung-Hwan,Kim. Seok-Joong,Shin. Sang-Tae,Cho.
간행물명
韓國受精卵移植學會誌
권/호정보
2014년|29권 1호|pp.59-66 (8 pages)
발행정보
한국수정란이식학회
파일정보
정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{mu}l$ and $50ng/{mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{mu}l$ than the $50ng/{mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.